Bottom Line:
Based on these results, OMP, a widely prescribed proton pump inhibitor known to target the ATP1A2 gene product, was chosen and tested.Sublethal doses of PZQ combined with OMP significantly increased worm mortality in vitro when compared with PZQ or OMP alone, thus evidencing a synergistic effect.We demonstrated the potential of this strategy by showing that PZQ in combination with OMP displayed increased efficiency against S. mansoni adult worms in vitro when compared with either drug alone.

Background: Treatment and morbidity control of schistosomiasis relies on a single drug, praziquantel (PZQ), and the selection of resistant worms under repeated treatment is a concern. Therefore, there is a pressing need to understand the molecular effects of PZQ on schistosomes and to investigate alternative or synergistic drugs against schistosomiasis.

Methodology: We used a custom-designed Schistosoma mansoni expression microarray to explore the effects of sublethal doses of PZQ on large-scale gene expression of adult paired males and females and unpaired mature females. We also assessed the efficacy of PZQ, omeprazole (OMP) or their combination against S. mansoni adult worms with a survival in vitro assay.

Principal findings: We identified sets of genes that were affected by PZQ in paired and unpaired mature females, however with opposite gene expression patterns (up-regulated in paired and down-regulated in unpaired mature females), indicating that PZQ effects are heavily influenced by the mating status. We also identified genes that were similarly affected by PZQ in males and females. Functional analyses of gene interaction networks were performed with parasite genes that were differentially expressed upon PZQ treatment, searching for proteins encoded by these genes whose human homologs are targets of different drugs used for other diseases. Based on these results, OMP, a widely prescribed proton pump inhibitor known to target the ATP1A2 gene product, was chosen and tested. Sublethal doses of PZQ combined with OMP significantly increased worm mortality in vitro when compared with PZQ or OMP alone, thus evidencing a synergistic effect.

Conclusions: Functional analysis of gene interaction networks is an important approach that can point to possible novel synergistic drug candidates. We demonstrated the potential of this strategy by showing that PZQ in combination with OMP displayed increased efficiency against S. mansoni adult worms in vitro when compared with either drug alone.

pntd.0004086.g006: Kaplan-Meier survival curves for adult S. mansoni worms treated in vitro with PZQ+OMP, PZQ or OMP.Adult worm couples were treated with a synergistic combination of PZQ+OMP (blue line), with PZQ alone (red line) or with OMP alone (black line). The assays were performed with paired adult worm couples (20 couples for each of the three conditions in each of the two biological replicates) and the fraction of surviving worms was recorded after 2, 24, 48, 72, 96 and 120 hours; the Kaplan-Meier survival curve was calculated using all the events of the two biological replicas together, and the Log-rank Mantel-Cox statistical test was used to calculate the significance of the Hazard Ratio between OMP+PZQ and PZQ alone. The effect of the synergistic combination was analyzed for each gender separately: (A) for scoring males percent survival, couples were incubated in the presence of 150 ng/ml PZQ, with or without 25 μg/ml OMP, or in the presence of 25 μg/ml OMP alone; and (B) for scoring females percent survival, couples were incubated in the presence of 532 ng/ml PZQ, with or without 25 μg/ml OMP, or in the presence of 25 μg/ml OMP alone.

Mentions:
To obtain a statistical estimate of the significance of the PZQ+OMP effect, we performed a Kaplan-Meier survival analysis of male or female adult worms treated with PZQ versus PZQ+OMP, using all the events of the two biological replicas in the analysis. Fig 6A shows the Kaplan-Meier curve of male survival, which revealed an 8-fold statistical increase in male death (p-value < 0.0001) for paired worms treated with the synergistic combination of a sublethal dose of PZQ (150 ng/ml) + 25 μg/ml OMP (Fig 6A, blue line), when compared with male death in worm pairs treated with the sublethal dose of PZQ alone (Fig 6A, red line). For the females survival assay, worm couples were treated with a synergistic combination of PZQ (532 ng/ml) + 25 μg/ml OMP, which caused a 2.98-fold statistical increase in female death (p-value = 0.005) (Fig 6B, blue line) when compared with the female death in worm pairs treated with PZQ (532 ng/ml) alone (Fig 6B, red line). In contrast, male and female worms remained viable until the end of the incubation period (120 h) in the groups treated with 25 μg/ml OMP alone (Fig 6A and 6B, black lines), as well as in the no-drugs negative control group.

pntd.0004086.g006: Kaplan-Meier survival curves for adult S. mansoni worms treated in vitro with PZQ+OMP, PZQ or OMP.Adult worm couples were treated with a synergistic combination of PZQ+OMP (blue line), with PZQ alone (red line) or with OMP alone (black line). The assays were performed with paired adult worm couples (20 couples for each of the three conditions in each of the two biological replicates) and the fraction of surviving worms was recorded after 2, 24, 48, 72, 96 and 120 hours; the Kaplan-Meier survival curve was calculated using all the events of the two biological replicas together, and the Log-rank Mantel-Cox statistical test was used to calculate the significance of the Hazard Ratio between OMP+PZQ and PZQ alone. The effect of the synergistic combination was analyzed for each gender separately: (A) for scoring males percent survival, couples were incubated in the presence of 150 ng/ml PZQ, with or without 25 μg/ml OMP, or in the presence of 25 μg/ml OMP alone; and (B) for scoring females percent survival, couples were incubated in the presence of 532 ng/ml PZQ, with or without 25 μg/ml OMP, or in the presence of 25 μg/ml OMP alone.

Mentions:
To obtain a statistical estimate of the significance of the PZQ+OMP effect, we performed a Kaplan-Meier survival analysis of male or female adult worms treated with PZQ versus PZQ+OMP, using all the events of the two biological replicas in the analysis. Fig 6A shows the Kaplan-Meier curve of male survival, which revealed an 8-fold statistical increase in male death (p-value < 0.0001) for paired worms treated with the synergistic combination of a sublethal dose of PZQ (150 ng/ml) + 25 μg/ml OMP (Fig 6A, blue line), when compared with male death in worm pairs treated with the sublethal dose of PZQ alone (Fig 6A, red line). For the females survival assay, worm couples were treated with a synergistic combination of PZQ (532 ng/ml) + 25 μg/ml OMP, which caused a 2.98-fold statistical increase in female death (p-value = 0.005) (Fig 6B, blue line) when compared with the female death in worm pairs treated with PZQ (532 ng/ml) alone (Fig 6B, red line). In contrast, male and female worms remained viable until the end of the incubation period (120 h) in the groups treated with 25 μg/ml OMP alone (Fig 6A and 6B, black lines), as well as in the no-drugs negative control group.

Bottom Line:
Based on these results, OMP, a widely prescribed proton pump inhibitor known to target the ATP1A2 gene product, was chosen and tested.Sublethal doses of PZQ combined with OMP significantly increased worm mortality in vitro when compared with PZQ or OMP alone, thus evidencing a synergistic effect.We demonstrated the potential of this strategy by showing that PZQ in combination with OMP displayed increased efficiency against S. mansoni adult worms in vitro when compared with either drug alone.

Background: Treatment and morbidity control of schistosomiasis relies on a single drug, praziquantel (PZQ), and the selection of resistant worms under repeated treatment is a concern. Therefore, there is a pressing need to understand the molecular effects of PZQ on schistosomes and to investigate alternative or synergistic drugs against schistosomiasis.

Methodology: We used a custom-designed Schistosoma mansoni expression microarray to explore the effects of sublethal doses of PZQ on large-scale gene expression of adult paired males and females and unpaired mature females. We also assessed the efficacy of PZQ, omeprazole (OMP) or their combination against S. mansoni adult worms with a survival in vitro assay.

Principal findings: We identified sets of genes that were affected by PZQ in paired and unpaired mature females, however with opposite gene expression patterns (up-regulated in paired and down-regulated in unpaired mature females), indicating that PZQ effects are heavily influenced by the mating status. We also identified genes that were similarly affected by PZQ in males and females. Functional analyses of gene interaction networks were performed with parasite genes that were differentially expressed upon PZQ treatment, searching for proteins encoded by these genes whose human homologs are targets of different drugs used for other diseases. Based on these results, OMP, a widely prescribed proton pump inhibitor known to target the ATP1A2 gene product, was chosen and tested. Sublethal doses of PZQ combined with OMP significantly increased worm mortality in vitro when compared with PZQ or OMP alone, thus evidencing a synergistic effect.

Conclusions: Functional analysis of gene interaction networks is an important approach that can point to possible novel synergistic drug candidates. We demonstrated the potential of this strategy by showing that PZQ in combination with OMP displayed increased efficiency against S. mansoni adult worms in vitro when compared with either drug alone.